COMPUTATIONAL CHARACTERIZATION FOR GENOME INTEGRATION SITES OF HEPATITIS B VIRUS (HBV) AND GENE TARGETS OF HBV X PROTEIN

Abstract

Our laboratory had enriched for HBV sequences in 48 HBV-associated HCC patients and employed the FLX Genome Sequencer to characterize variations in the HBV DNA as well as HBV integration events in these patients. In this thesis, I employed a computational workflow to analyze the high-throughput sequencing data, and identified 60 contigs/reads with altered HBV DNA and 63 contigs/reads carrying both HBV and human DNA within the same read from which the HBV-HG junction sites were inferred. Interestingly, the HBV-HG integrations were found to preferentially occur at the HBx gene locus (27/63=42.9%) and the 3¿ C-terminal of HBx carrying p53 binding domain was often deleted to fuse with the human genome. Deletion of p53 binding domain of HBx may potentially promote carcinogenesis in HCC patients, as p53 is a well-known tumor suppressor. The N-terminal two third of HBx gene carrying transactivation domains were often retained in the integrated form. Significantly, our laboratory has successfully experimentally validated a subset of the integrated sequences and the expression of chimeric transcripts. By characterization of HBV genome integration sites using high throughput targeted genome sequencing, we are now better positioned to gain improved insights on how HBV genome integration may contribute to hepatocarcinogenesis in HCC patients. To further elucidate the role of the HBx gene in HCC, our laboratory employed chromatin immunoprecipitation and sequencing using the Solexa Genome Sequencer (ChIP-Seq) on immortalized liver cell line, THLE3 using HBx antibodies. I employed a computational workflow to integrate the high throughput ChIP-Seq data, microarray expression profiles for both cell lines (THLE3) and 100 HBV-associated HCC patients, and the clinical data of the 100 HCC patients. 143 potential HBx deregulated direct gene targets were identified in THLE3 cells, indicating the pleiotropic nature of HBx: interact with a variety of transcription factors and deregulate a large set of genes. 18 of these 143 HBx-associated deregulated genes were also consistently differentially deregulated in the 100 HCC patients. Seven of these 18 genes were found significantly associated with various patients¿ clinical features including survival, tumor grade, tumor invasion, liver cirrhosis, tumor capsulation and multifocality. By identification of clinically associated potential HBx deregulated direct gene targets, we are now in a better position to explore the role of HBx in hepatocarcinogenesis in HCC patients.